The yeast protein Dna2, originated from the budding yeast (Saccharomyces cerevisiae), is well known as an enzyme having a 5′-3′ DNA helicase activity and a structure-specific endonuclease activity. The properties of Dna2 have been recently reported in detail (S. Bae, K. Bae, J. Kim, and Y. Seo, “RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes” (2001) Nature, 412, 456-461).
Dna2 is an essential enzyme in DNA replication and repair, and specifically plays a roll in unwinding a double helix into separate single strands during DNA replication initiation, maturating Okazaki fragments during lagging strand replication, and repairing double-strand breaks. While various DNA helicases including Dna2 have been found in viruses, bacteria and eukaryotes, most of them are originated from mesophiles. Thus, such DNA helicases are rapidly deactivated under conditions causing heat-denaturation of double-stranded DNA.
If a heat-stable enzyme having a 5′-3′ DNA helicase activity is discovered, all processes involving heat-denaturation of double-stranded DNA at 94° C. or greater, rapid cooling of the heat-denatured double-stranded DNA and an enzymatic reaction could be carried out in one tube. Such a heat-stable enzyme would open the way for establishing a combinational experimental system of a DNA replication-repair conjugation system and a DNA amplification reaction (PCR) or the like, and in the development of new in vitro gene replication or mutation methods. Thus, there is a strong need for an enzyme capable of unwinding a double helix into separate single strands during DNA replication initiation, maturating Okazaki fragments during lagging strand replication, and repairing double-strand breaks, even under stringent conditions.